Multi-drug resistant bacteria affect 2 million people in the U.S. every year, resulting in 23,000 deaths and causing an economic burden of an estimated $20 billion. The problem is exacerbated by the fact that development of new antibacterial agents has slowed down in the past decade. In short, bacteria are gaining resistance to available antibiotics at a rate faster than new antibiotics can be developed and brought to market.
In order to impede the takeover by resistant strains and to preserve the existing antibiotics, physicians must improve the process of prescribing antibiotics. Ideally, an antibiotic therapy should start only after confirming the susceptibility of the infecting bacteria to the antibiotic. However, physicians typically treat serious infections empirically by prescribing broad-spectrum antibiotics, because standard antibiotic susceptibility tests (ASTs) require long cell culturing steps. An antibiotic susceptibility test (AST) determines whether or not bacterial isolates from a patient's blood, wound specimens, or urine are susceptible to administered antibiotics. One of the gold standard tests, a broth dilution test, is performed by preparing a set of bacteria solutions, which are incubated overnight in the presence of different antibiotics with different concentrations. If the bacteria are resistant to the antibiotic, they multiply and the solution eventually becomes turbid. The limits of a typical optical measurement require the bacteria solution to be incubated for 16-20 hours. Similarly, the disk diffusion method is performed by applying a bacterial inoculum to the surface of an agar plate, on which antibiotic disks are placed. After incubation for 16-24 hours, the antibiotic inhibits the bacteria growth in the regions where it diffuses. The susceptibility of the bacterial strain can be determined from the size of the bacteria-free zones around the antibiotic disks.
However, both these tests have a shared shortcoming: one has to wait long enough such that the population reaches minimum detectable growth levels. Accordingly, there exists in the art a need for novel, rapid, sensitive and robust methods to determine the antibiotic susceptibility of bacteria.
It is clear that the above-described mainstay methods have limitations, and multi-drug resistant bacteria pose a grand challenge in global healthcare. Hence, the development of rapid antibiotic susceptibility tests (ASTs) is an active research area, with many publications and patents. The focus is on observing bacterial resistance at early stages of cell growth. Polymerase chain reaction (PCR) is the quintessential genotypic method. PCR directly detects the resistance gene of a very small bacterial sample and can provide fast identification of antibiotic resistance. However, it has limited utility, because only a few resistance genes are firmly associated with phenotypic antibiotic resistance. There are simply too many genetic mutations, acquisitions, and expressions to be routinely identified by current PCR techniques. Further, the use of PCR at point-of-care settings remains challenging.
Phenotypic methods, on the other hand, are based on observable characteristics of bacteria. Novel phenotypic ASTs typically employ microfluidics and microdevices, because these devices allow for effective use of samples and multiplexing. Researchers have been exploring different approaches based on microfluidics. In the first type of experiments, the growth of bacteria was directly observed in small volumes (e.g., microfluidic channels) in order to determine susceptibility to different antibiotics. This approach was pushed down to the single cell limit by confining cells in drug-infused nano and pico-liter droplets. In another approach, bacteria were adhered to oscillating microstructures, such as microcantilevers or magnetic microbeads, under administered antibiotics; here, the oscillation frequency decreased due to added bacteria mass if the bacteria multiplied, indicating resistance. Finally, measuring the physical or chemical properties of a medium (e.g., changes in electrical impedance or pH) due to bacterial proliferation allowed for measuring microbial growth. The changes with administered antibiotics then provided the desired susceptibility testing.
While most of the above-mentioned techniques are ingenious, very few have found their ways into the mainstream. There are still several major challenges. Some techniques require delicate microscopy or are too complicated to be implemented at point-of-care settings. Others rely upon labeling (e.g., fluorescent labeling), which limits utility. If the test determines susceptibility based on whether or not the bacteria are growing, the heterogeneity of the antimicrobial response of bacteria becomes an issue. In summary, there is a significant need for new approaches.